Zinc is an essential nutrient for all cells due to its key role as a catalytic and structural cofactor. Competition for zinc and other nutrient metal ions is critical for determining the outcome of host- pathogen interactions. Metalloregulatory proteins sense both zinc deficiency and excess and are required for bacterial acclimation to changes in zinc availability. In Bacillus subtilis, a Gram positive model organism, zinc sufficiency is sensed by Zur (zinc uptake repressor) and zinc excess by CzrA. When zinc levels are limiting for growth, Zur repression is relieved resulting it increased zinc uptake (mediated by the ZnuABC transporter), Zn mobilization by displacement of small, ribosome-associated zinc proteins (L31 and L33), and replacement of a key zinc dependent enzyme (FolE) by a non-zinc requiring alternate enzyme. In the next project period, we will investigate the detailed molecular mechanisms of zinc sensing and define the major components of the labile zinc pool in the cell. Preliminary results have led to five core hypotheses that form the foundation of the Specific Aims. Aim 1a will address the hypothesis that regulation by Zur involves a graded response as the cell transitions from zinc sufficiency to deficiency, and will test the role of negative cooperativity in zinc-binding to Zur in expanding the range of zinc responsiveness. In Aim 1b, we will adapt genetically encoded fluorescent reporters to monitor free zinc levels in vivo and seek to reconcile the exceptionally high (sub-picomolar) zinc affinity of the Zur and CzrA sensors with emerging evidence consistent with free zinc levels in the mid-picomolar range. Aim 2 will address the role of the low molecular weight thiol bacillithiol (BSH) as the major buffer of the labile zinc pool (Aim 2a), the role of small, dispensable ribosome-associated proteins as the major storage form of zinc (Aim 2b), and the roles of these two zinc pools in metallation of FolE, a zinc metalloenzyme sensitive to zinc availability, and other key zinc enzymes. Limitation for zinc by calprotectin, and likely other factors, is a key part of the human innate immune response. Since all of the key zinc homeostasis factors described here are conserved in many important human pathogens, these studies will provide key background information to facilitate the ultimate development of antimicrobial agents that affect metal ion homeostasis.